The present invention is a continuation-in-part of an application entitled Cutaneous Methods of Measuring Body Substances, Ser. No. 63,159, filed Aug. 2, 1979 which is now pending. This application disclosed the concept of analyzing lactate by reacting lactate with lactic oxidase to produce hydrogen peroxide. The hydrogen peroxide was then measured polarographically using a cutaneous electrode. The present invention deals with the problem of rapidly measuring lactic acid or lactate in whole mammalian blood.
Medical science is now realizing the importance of rapidly measuring lactic acid levels in blood, skeletal muscles and the heart. Lactic acid levels in blood appears to be an indication of certain critical features in mammals. A high blood lactic acid level frequently is an indication that a mammal is about to go into shock. For accident victims, it could be extremely critical to rapidly determine the lactic acid level. Such a determination should be rapid and use minute quantities of blood in order to permit repeated measurements of lactic acid level. In infants, lactic acid levels are important indicators of defects in metabolism of carbohydrates. With infants, sample sze is extremely critical since the amount of blood in the infant is substantially less than that of an adult.
Some authorities theorize that the ratio of lactic acid to pyruvic acid in blood is important. Accordingly, any method of measuring lactic acid should preferably also enable one to measure pyruvic acid in order to determine this ratio.
Currently, there are various methods to measure lactic acid. When the lactic acid is measured in whole blood, the plasma generally must be separated from the blood to eliminate undesirable substances which could cause side reactions. For example, the lactic acid has been measured indirectly by reduction using a lactic dehydrogenase which consumes oxygen and the oxygen decrease is thereby measured to indicate lactic acid levels. This is an indirect measurement of lactic acid and tends to be extremely expensive and time consuming. In these reactions, the blood is separated from the lactic dehydrogenase by using a semi-permeable membrane. Other methods include colorimetric methods in which the blood plasma is separated from the whole blood cells using filtration or centrifugation to separate the whole blood cells from the plasma. The plasma can then be reacted with lactic oxidase to produce hydrogen peroxide which can be colorimetrically measured. These methods are time consuming and expensive and fail to provide a means to rapidly detect lactic acid directly.
Furthermore, these methods fail to provide a means to measure the ratio of lactic acid to pyruvic acid and further fail in that they do not provide a means to measure in situ lactic acid levels. This would be particularly important in measuring lactic acid levels in the heart.
About 15 years ago, enzyme-coupled electrodes were reported for the polarographic analysis of the substances. For example, my U.S. Pat. No. 3,539,455 discloses a membrane polarographic electrode system method for the rapid and accurate quantitative analysis of substances which theretofor posed difficulties in analyzing directly by polarographic methods. According to the description in my mentioned patent, small molecular substances, such as glucose, were measured with a membrane polarographic electrode system. By use of cellulose or another membrane which is permeable to small molecules such as glucose, but impermeable to proteins, the membrane keeps glucose oxidase enzyme on the side of the membrane with the anode for reaction with glucose. Therefore, for example, when a sample of blood were placed on the membrane side opposite the electrode with an aqueous solution of enzyme and oxygen on the electrode side of the membrane, low molecular weight molecules such as glucose pass from the blood samples through the membrane for enzymatic reaction adjacent the electrode. After a certain period of time, a steady state is reached when the hydrogen peroxide concentration is directly proportional to the glucose concentration and the cell produces a current flow as a function of the amount of hydrogen peroxide being formed which serves as an indication of the amount of glucose present.
Lactic oxidase for some time has been puzzling the scientific world. For some time, it was a matter of dispute whether lactic oxidase, in fact, could produce hydrogen peroxide from lactic acid. One possible reason for this dispute is frequently the enzyme had associated therewith a substantial amount of a catalase which would quickly consume hydrogen peroxide. A second source of this dispute was the improper characterization of lactic oxidase. Certain enzymes, which were actually lactic oxoreductases, were characterized as lactic oxidases. These lactic oxoreductases, instead of producing hydrogen peroxide and pyruvate from lactic acid, produced acetic acid, carbon dioxide and water. However, it has been recently appreciated in the literature that there are true lactic oxidases and which are substantially catalase free and suitable for the reaction to produce hydrogen peroxide from lactic acid or lactate. Such an enzyme is disclosed in U.S. Pat. No. 4,166,763, assigned to Eastman Kodak Company. This patent discloses an enzyme for use in analysis of lactic acid whereby the lactic acid is oxidized to produce pyruvate and hydrogen peroxide. The hydrogen peroxide is measured colorimetrically.